An in vitro DNA replication system of yeast 2 mum and ARS (autonomously-replicating-sequence) plasmid DNAs, developed in this laboratory several years ago, has been used to investigate the mechanism of DNA replication in eucaryotes. To identify and purify enzymes and components required for yeast DNA replication, the crude-extract system has been fractionated and reconstituted. Recently, two additional activities have been identified and purified: DNA topoisomerase I and ARS-binding protein(s). Independently, another group has shown that yeast DNA topoisomerase I is required for in vivo yeast DNA replication and for rRNA synthesis. This is consistent with our observation in vitro, further strengthening the usefulness of our in vitro system. We have also shown that the dnal-1 mutant is deficient in the initiation of chromosomal DNA replication. To aid in overproducing and purifying DNA1 protein, the DNA1 gene has been cloned, its nucleotide sequence determined and its regulation studied. Finally, two additional single-stranded DNA binding proteins (SSBs) (35KD and 45KD) have been purified from yeast crude extract, studied biochemically and their monospecific polyclonal antibodies raised from rabbits. The antibodies to these and to previously purified SSBs (14KD, 20KD, 26KD, and 38KD) have shown that the 38KD and 42KD and the 20KD and 26KD SSBs are immunologically related but the 14KD and 35KD SSBs are unique species. The antibodies have also been used to localize the 35KD, 38/42KD and 20KD/26KD SSBs to nuclei by immunofluoresence. The 35KD SSB is particularly interesting and important because it is recognized by an antiserum that reacts with mammalian PCNA/cyclin, a 36KD protein under cell-cycle control and thought to be involved in cellular DNA replication.